The goal of this Phase I project is to develop a highly efficient cell based assay technology that can be used to make antimicrobial discovery a much more efficient process. The technology would permit the large scale fingerprinting of any antimicrobial discovered in a natural product or developed by chemical synthesis. The technology would enable pharmaceutical R&D programs to create a comprehensive, expandable database that would be used as a standard reference for antimicrobial identification. This database would, for each antimicrobial, 1) indicate the uniqueness of its biological action, 2) provide information about its inhibitory mode of action (MOA), and 3) provide synergy/antagonism data for combinations with well known antibiotics. Individual antimicrobial programs could adopt this technology and database to add fingerprints from their own proprietary chemical libraries. The long-term objective would be to create a novel anti-infective identification technology that would have many applications. Natural product screens for antibiotics would use this technology to classify their "hits" to determine their novelty. Leads undergoing chemical modification to increase potency would be screened using our technology to ensure each derivative inhibits the same essential cellular target in vivo, thus guiding and validating structure activity relationships (SARs). With this technology, the MOA of a novel antibiotic may be inferred and might even be proven if its fingerprint matches that of a strain whose essential protein can be down regulated. We are facing a critical need for novel antimicrobials. Drug resistance and even multi-drug resistance continue to emerge in important human pathogens (e.g., Staphylococcus aureus, Mycobacterium tuberculosis). Added to this is the newly realized bio-terrorism threat in which microbes, intentionally engineered to be multiply antibiotic resistant, can be spread throughout the populace. The high expense and low rate of antibiotic discovery in current R&D programs, unfortunately, is a major obstacle preventing many companies from having competitive antimicrobial programs. A major bottleneck in antimicrobial discovery is in assessing chemical libraries to efficiently focus in on the most promising new candidates. Funding of this project will help to develop a technology that can assist all antimicrobial development projects in their efforts to efficiently discover novel antibiotics.